Scalable method for producing transfection reagents
The invention provides easy-to-scale methods of creating DNA/transfection reagent master mixes for transfecting cells in culture.
1 . A method of preparing a transfection master mix comprising the steps of:
(1) introducing a DNA solution and a transfection reagent solution into a mixing container to create a transfection master mix, wherein the volume of the transfection master mix is 1 L or greater;
(2) rocking the mixing container with a mechanical rocking device for a period of about 1 minute to about 10 minutes at 10 rpm to 15 rpm and about 7 degrees to about 12 degrees rocking angle to reach a steady state concentration of DNA within the transfection master mix; and
(3) incubating the transfection master mix for an incubation period of about 20 minutes to about 30 minutes, wherein the transfection master mix is substantially still during the incubation period;
wherein the transfection master mix consists essentially of the DNA solution and the transfection reagent solution;
wherein the DNA solution comprises plasmids selected from the group consisting of recombinant adeno-associated virus (rAAV) cis plasmids, rAAV trans-rep-cap plasmids, and helper plasmids; and
wherein the transfection master mix is for co-transfection of rAAV packaging cells for rAAV production.
2 . The method of claim 1 , wherein the mixing container is (a) a single-use disposable container; (b) a pre-sterilized container; (c) a plastic bag; or (d) a single-use cell culture bag.
3 . The method of claim 2 , wherein the mixing container is a plastic bag or a single-use cell culture bag, which bag is fully or partially inflated.
4 . The method of claim 1 , wherein the transfection reagent solution comprises a chemical transfection reagent selected from the group consisting of: a cationic polymer, calcium phosphate, DEAE-dextran, an activated dendrimer, and a cationic lipid.
5 . The method of claim 4 , wherein the cationic polymer is polyethylenimine.
6 . The method of claim 1 , wherein the DNA solution and/or the transfection reagent solution is sterile.
7 . The method of claim 1 further comprising the step of (a) sterilizing the DNA solution prior to introducing the DNA solution into the mixing container;
and/or (b) sterilizing the transfection reagent solution prior to introducing the transfection reagent solution into the mixing container.
8 . The method of claim 1 , wherein the mechanical rocking device comprises a rocking platform.
9 . The method of claim 1 , wherein the volume of the transfection master mix is 3 L or greater, 12 L or greater, 15 L or greater, 25 L or greater, 50 L or greater, or 100 L or greater.
10 . The method of claim 1 , wherein the volume of the transfection master mix is between 1 L and 100 L.
11 . The method of claim 10 , wherein the volume of the transfection master mix is 1 L, 3 L, 12 L, 15 L, 25 L, or 29 L.
12 . The method of claim 1 , wherein (a) the volume of the transfection master mix is 4 L, and the rocking the mixing container is at an angle of 7 degrees, at 10 rpm, and for about 1 minute to about 2 minutes; or (b) the volume of the transfection master mix is 12 L, and the rocking the mixing container is at an angle of 12 degrees, at 15 rpm, and for about 1 minute to about 2 minutes.
13 . The method of claim 1 , wherein the step of introducing the DNA solution and the transfection reagent solution into the mixing container is accomplished sterilely.